Project Summary/Abstract HIV initially assembles as a non-infectious, immature particle that must undergo a maturation process to become infectious. Assembly and maturation involve large-scale rearrangements of the viral capsid. These large-scale rearrangements, in turn, are associated with programmed local conformational changes in capsid protein segments termed molecular switches. In these studies, we will use structural, biophysical, and other complementary approaches to understand the structure of the immature HIV-1 capsid and how a molecular switch spanning the CA-SP1 region of the HIV-1 Gag protein controls immature virus assembly and maturation. We propose to: (1) Test the hypothesis that the CA-SP1 junction folds into a 6-helix bundle that undergoes reversible and cooperative unfolding and folding within immature virions to regulate access of the viral protease during maturation. We will also determine how small molecule maturation inhibitors like bevirimat disrupt this process by binding and stabilizing the 6-helix bundle. We will also initiate experiments to understand how Gag-Gag interactions change during the early stages of maturation. (2) Elucidate the detailed molecular organization of the immature HIV-1 Gag lattice. In particular, we will determine the high resolution details of how the outermost set of Gag-Gag interactions (mediated by the CA-NTD domain) are organized within the immature particle. We will also test the hypotheses that interactions between the CA-NTD layer and the underlying CA-CTD layer are important for proper assembly and may be part of a structural network that links two molecular switches that flank the CA region. Finally, we will also analyze the immature Gag lattices of other retroviruses, in order to elucidate the general nature of switch-mediated Gag assembly. If successful, these studies will lead to a thorough understanding of the immature capsid structure of retroviruses, how local conformational transitions translate into large-scale capsid rearragenements during retroviral assembly and maturation, and how small molecule inhibitors may be deployed to disrupt capsid rearrangement.